CAJ | 학술논문

目的建立荧光核酸染料PicoGreen用于循环肿瘤DNA定量的方法并以乳腺癌为例初步探讨其在肿瘤辅助诊断中的作用. 方法采用QIAamp DNA blood kit提取血清中的循环肿瘤DNA并分析其提取效率,分析PicoGreen荧光分光光度法对微量DNA进行精确定量的范围和可靠性.以受试者工作特征曲线(ROC)判断循环肿瘤DNA应用于乳腺癌辅助诊断的可行性. 结果 QIAamp DNA blood kit提取血清中DNA的效率在37.8%～46.2%,平均43.4%.用PicoGreen荧光法,可检出低至1 ng的循环肿瘤DNA,并且在1～500 ng范围内呈现良好的直线关系.用此法检测乳腺癌组血清DNA浓度为(169.70±124.10)μg/L,对照组乳腺良性病变为(51.70±29.04)μg/L,健康人为(54.30±36.84)μg/L.经Mann-Whitney检验二者差异有统计学意义.ROC下面积为0.899(95%CI为0.848～0.951),当DNA诊断界值为96.0/μg/L时,灵敏度为78.2%,特异度为90.0%,提示血清DNA浓度有较好的诊断应用价值. 结论应用PicoGreen荧光分光光度法可有效检测微量的血清循环肿瘤DNA,且乳腺癌组循环肿瘤DNA浓度明显高于对照组,提示循环肿瘤DNA为有价值的乳腺癌辅助诊断指标之一.
목적 건립형광핵산염료PicoGreen용우순배종류DNA정량적방법병이유선암위례초보탐토기재종류보조진단중적작용. 방법 채용QIAamp DNA blood kit제취혈청중적순배종류DNA병분석기제취효솔,분석PicoGreen형광분광광도법대미량DNA진행정학정량적범위화가고성.이수시자공작특정곡선(ROC)판단순배종류DNA응용우유선암보조진단적가행성. 결과 QIAamp DNA blood kit제취혈청중DNA적효솔재37.8%～46.2%,평균43.4%.용PicoGreen형광법,가검출저지1 ng적순배종류DNA,병차재1～500 ng범위내정현량호적직선관계.용차법검측유선암조혈청DNA농도위(169.70±124.10)μg/L,대조조유선량성병변위(51.70±29.04)μg/L,건강인위(54.30±36.84)μg/L.경Mann-Whitney검험이자차이유통계학의의.ROC하면적위0.899(95%CI위0.848～0.951),당DNA진단계치위96.0/μg/L시,령민도위78.2%,특이도위90.0%,제시혈청DNA농도유교호적진단응용개치. 결론 응용PicoGreen형광분광광도법가유효검측미량적혈청순배종류DNA,차유선암조순배종류DNA농도명현고우대조조,제시순배종류DNA위유개치적유선암보조진단지표지일.
Objective To establish the methods for quantitating the circulating tumor DNA with PicoGreen fluorescent nucleic acid stain and investigate the role of the quantitative analysis in diagnosis of breast cancer. Methods Circulating tumor DNA was isolated from serum with QIAmp blood kit and was quantitated by spectrofluorometry with PicoGreen fluorescent stain. Receiver operator characteristic ( ROC) curve and area under the curve were used to estimate the role of DNA quantification in diagnosis of breast cancer. Results The efficiency of QIAamp blood kit isolating DNA from serum was 37. 8%-46. 2%, average 43. 4%. Circulating tumor DNA concentration as low as 1 ng could be detected by PicoGreen spectrofluorometry. and the detected range was 1-500 ng/0. 2 ml. The median concentration of serum DNA in breast cancer group was (169. 70+ 124. 10) μg/L, and that of healthy control and breast benign group were (54. 30±36. 84) μg/L and (51. 70±29. 04) μg/L, respectively (P<0. 01). The area under the ROC curve was 0. 899 (95% CI: 0.848-0.951), and the sensitivity was 78. 2%, the specificity was 90% by using the cutoff value of 96. 0 ng/ml. Conclusions The concentration of circulating tumor DNA can be efficiently quantitated by PicoGreen spectrofluorometry, which indicates the potential of clinical applicability in breast cancer diagnosis.